Identification of gene expression changes in human cancer using bioinformatic approaches
Washington University School of Medicine
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Discovering biomarkers and molecular drivers of head and neck endocrine tumors was the inspiration for this thesis. Here, I describe the molecular evaluation of tumors of the thyroid and parathyroid endocrine glands for the purpose of identifying somatic driver alterations in these cancers. While molecular interplay of the germline genomic background of an individual and the somatic genome that emerges throughout the lifetime plays significant roles in increasing the susceptibility to cancer and in driving the malignant phenotype, the major known contributors to cancer remain the acquired somatic mutations. Analysis of a sporadic and recurring parathyroid carcinoma, with incidence of 1 per million population, revealed mutations in mTOR, MLL2, CDKN2C and PIK3CA and comparison of patient-matched primary and recurrent malignant tumors uncovered loss of PIK3CA activating mutation during the evolution of the tumor. Loss of the short arm of chromosome 1 along with somatic missense and truncating mutations in CDKN2C and THRAP3 provided new evidence for the potential role of these as tumor suppressors. Hürthle cell thyroid carcinoma accounts for a small proportion of all thyroid cancers; however, this malignancy often presents at an advanced stage and poses unique challenges. Genomic analysis revealed large regions of copy number variation encompassing nearly the entire genomes accompanied also by near haploidization. Moreover, I identified loss-of-function mutations of the tumor suppressor gene MEN1 in 4% of patients. Repeated alterations of the epigenetic machinery in anaplastic thyroid carcinoma, one of the most fatal of all adult solid malignancies, and novel gene fusions including MKRN1-BRAF, FGFR2-OGDH and SS18-SLC5A11 are reported here. The transcriptomic analysis suggested known drug targets such as FGFRs, VEGFRs, KIT and RET to have low expressions in this cancer; however, through integrative data analysis, I identified the mTOR signaling pathway as a potential therapeutic target for anaplastic thyroid cancer. Molecular analysis of papillary thyroid carcinoma and benign thyroid nodules revealed very low mutation rates in these tumors with CYP1B1, PTPRE, CTSH and RUNX1 emerging as promising diagnostic markers. The key somatic mutations identified in these studies can serve as novel diagnostic markers as well as therapeutic targets.
No abstract available.
Repositioning existing drugs for new therapeutic uses is an efficient approach to drugdiscovery. However, most successful repositioning cases to date have been serendipitous; thegoal of my thesis was to use computational methods to rationally discover drug repositioningcandidates.I first virtually screened (VS) 4621 drugs against 252 drug targets with molecular docking.This method emphasized removing potential false positives using stringent criteria fromknown interaction docking, consensus scores, and rank information. Published literatureindicated experimental evidence for 31 top predicted interactions, supporting the approach.The chemotherapeutic nilotinib was validated as a potent MAPK14 inhibitor in vitro (IC5040nM), suggesting a potential use in inflammatory diseases.I then applied this method to the cancer target EGFR, predicting the anti-HIV drug tenofovirdisoproxil fumarate (TDF) as a novel inhibitor. In vitro, TDF inhibited the proliferation andEGFR-signaling of an EGFR-overexpressing cell line, but did not inhibit EGFR in directkinase binding assays. This study highlighted limitations of computational and experimentalmethodologies that should be considered when interpreting or designing other studies.We then screened 1,120 off-patent drugs against the triple-negative breast cancer (TNBC)target p90RSK using both VS and high-throughput (HTS) methods. VS predicted a set ofcompounds 26-times enriched for known RSK inhibitors and 11 times enriched for HTS hits,underscoring its efficiency. In secondary screens, the chemotherapeutic ellipticine and thebioflavonoids luteolin and apigenin inhibited RSK activity (IC50 0.50-4.77μM), blocked RSKsignaling, and inhibited TNBC cell proliferation. These drugs thus have potential to berepositioned to TNBC.Finally, we rationally repositioned renal cell carcinoma drugs for a patient with a rare tongueadenocarcinoma. Whole genome and transcriptome sequencing of the patient’s tumor andnormal cells detected sequence, copy number, and expression aberrations, and analysis suggested that the tumor was driven by the RET oncogene. Treatment with RET-inhibitingdrugs stabilized the disease for eight months, after which the disease progressed. We alsosequenced the post-treatment tumor and found changes consistent with acquired therapeuticresistance.Overall, this thesis details two novel high-throughput approaches for drug repositioning:virtual screening of drugs and targets and personalized medicine via sequencing.
No abstract available.
The thyroid gland, necessary for normal human growth and development, is essential for the regulation of metabolism. Its function – to produce and secrete appropriate levels of thyroid hormone – is simple; however accurate assessment of thyroid abnormality is challenging and a fundamental understanding of the normal thyroid is therefore needed. One way to characterize the normal functioning of the thyroid gland is to study the epigenome and resulting transcriptome within its constituent cells. In this study, we compare the consistency of chromatin state annotations across the epigenomes from the grossly uninvolved tumour-adjacent thyroid tissue of four human individuals using ChIP-seq and RNA-seq. We profile four activating (H3K4me1, H3K4me3, H3K27ac, H3K36me3) and two repressing (H3K9me3, H3K27me3) histone modifications, identify chromatin states using a hidden Markov model, produce a novel metric for model selection, and establish epigenomic maps of 19 chromatin states. We found that epigenetic features characterizing promoters and transcription elongation tend to more consistent across epigenomes and that epigenetically active genes consistent across all epigenomes tend to have higher expression than those that are not marked as epigenetically active in all samples. We also identified a set of 18 genes epigenetically active and consistently expressed in the thyroid that are likely relevant to thyroid function. Altogether, we believe the epigenomes presented in this work represent a useful resource to gain a deeper understanding of the underlying molecular biology of thyroid function and provide contextual information of thyroid and human epigenomic data for comparison and integration into future studies.
Retrieving relevant scientific papers in a scalable way is increasingly important, as more and more studies are published. PubMed’s relevant article recommendation is based on MeSH assignments by indexers, which requires significant human resources and can become a limitation in making papers searchable. Many recommendation systems use singular value decomposition (SVD) to pre-compute related products. In this study, we look at using latent semantic analysis (LSA), an application of SVD to determine relationships in a set of documents and terms, to find related biomedical papers. We focused on determining the best parameters for SVD in retrieving relevant biomedical articles given a paper of interest. Using PubMed's recommendations as guidance, we found that using cosine distance to measure document similarity leads to better results than using Euclidean distance. We re-evaluated other parameters, including the weighting scheme and the number of singular values and using a larger abstract corpus. Finally, we asked people to compare the relevant abstract retrieved with our method against those retrieved by PubMed. Our method retrieved sensible articles that were chosen over PubMed's relevant papers one-third of the time. We looked into the abstracts retrieved by either method and discuss possible areas for experimentation and improvement.
The immune system plays a critical role in cancer prevention and development. The stimulation of natural immune reaction in a cancer patient by adoptive T-cell therapy has shown success in treating metastatic melanomas and renal cell carcinomas. However, the use of adoptive T-cell therapy remains limited due to unpredictable outcomes and low response rates. In particular, adoptive T-cell therapy for breast cancer has not been realized, despite of the presence of immunogenic antigens such as over-expressed HER2, present in 20-40% of breast tumours. Using a unique transgenic mouse model, the global profiles of gene expression, miRNA abundance and single nucleotide variants (SNVs) were investigated to identify the molecular difference of murine mammary tumours with isogenic background, which exhibited complete regression (CR), partial regression (PR) or progressive disease (PD) outcome of adoptive T-cell therapy. The bioinformatics analyses were further carried out to identify uniquely activated pathways, prognostic gene expression signatures, the effect of post-transcriptional gene regulation and mutated genes unique to tumours with specific outcome. The largest differences in gene expression, miRNA and SNV profiles were repeatedly observed between the regressing (CR, PR) and non-regressing (PD) tumours, supporting the attribution of molecular differences to the immunotherapy outcome. In particular, the gene expression signatures derived from genes in immune-related pathways were experimentally validated to be strong prognostic markers for predicting the CR outcome. Comparison with the human breast cancer subtypes further revealed similarities of the non-regressing tumours with the basal subtype, and the regressing tumours with the HER2 subtype. The difference in miRNA profiles between CR and PR tumours suggested potential translational activities unique to PR, which was nearly identical to CR at the transcriptome level. The findings from this study show that tumour-derivied factors that either promote or suppress the immune system are responsible for the varying outcome of immunotherapy, and that the molecular characteristics can be further applied for the development of clinical prognostic tools, cancer vaccines and drug targets to enhance the efficacy of adoptive T-cell therapy.